: The Aer protein mediates aerotactic behavior in Escherichia coli. A growing list of other bacteria, including pathogens such as Vibrio cholerae and Yersinia pestis, have Aer homologs. The long-range goals of this project are to understand in molecular detail how the Aer protein senses changes in environmental oxygen levels and how that stimulus information is transmitted through the Aer molecule to control the cell's swimming movements. Aer appears to be located predominantly in the cytoplasm, but anchored to the inner face of the cytoplasmic membrane through a central segment of hydrophobic amino acids. To explore the role of membrane association in Aer function, the topology of the native molecule will be probed by accessibility to proteases and aqueous sulfhydryl modification reagents. Mutant proteins with deletions or substitutions in the hydrophobic segment will be tested for membrane association and aerotactic signaling ability in attempts to develop a soluble, active form of Aer. To identify conformational features that might play a role in Aer signal transduction, an extensive set of mutant proteins with single cysteine reporter residues will be constructed and used to examine intra- and intersubunit interactions between different regions of the native Aer molecule. Mutations that "lock" Aer into a stimulus-insensitive or stimulus-mimicked state will be isolated and used to trace the path of signal transmission through the molecule. Preliminary studies have established that the N-terminus of Aer binds flavin adenine dinucleotide (FAD), which might serve as a prosthetic group to monitor the redox state of an electron transport component. To test the redox-sensing model, in vitro assays of Aer signaling activity will be developed. The redox potential of FAD in native Aer will also be determined to identify electron transport components that could conceivably interact with Aer during stimulus detection. These studies promise to shed new light on biological mechanisms of oxygen-sensing. Moreover, the existence of Aer homologs in pathogenic bacteria suggests that aerotactic behavior might play a role in virulence. Thus, a molecular understanding of Aer signal transduction could lead to new anti-infection agents.